Rapid-Read Gated Amperometry

ABSTRACT

A sensor system, device, and methods for determining the concentration of an analyte in a sample is described. Input signals including multiple duty cycles of sequential excitation pulses and relaxations are input to the sample. One or more signals output from the sample within 300 ms of the input of an excitation pulse may be correlated with the analyte concentration of the sample to improve the accuracy and/or precision of the analysis. Determining the analyte concentration of the sample from these rapidly measured output values may reduce analysis errors arising from the hematocrit effect, mediator background, and other error sources.

REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/012,729 entitled “Rapid-Read Gated Amperometry” as filed on Dec. 10,2007, which is incorporated herein by reference.

BACKGROUND

Biosensors provide an analysis of a biological fluid, such as wholeblood, serum, plasma, urine, saliva, interstitial, or intracellularfluid. Typically, biosensors have a measurement device that analyzes asample residing in a sensor strip. The sample usually is in liquid formand in addition to being a biological fluid, may be the derivative of abiological fluid, such as an extract, a dilution, a filtrate, or areconstituted precipitate. The analysis performed by the biosensordetermines the presence and/or concentration of one or more analytes,such as alcohol, glucose, uric acid, lactate, cholesterol, bilirubin,free fatty acids, triglycerides, proteins, ketones, phenylalanine orenzymes, in the biological fluid. The analysis may be useful in thediagnosis and treatment of physiological abnormalities. For example, adiabetic individual may use a biosensor to determine the glucose levelin whole blood for adjustments to diet and/or medication.

Biosensors may be designed to analyze one or more analytes and may usedifferent sample volumes. Some biosensors may analyze a single drop ofwhole blood, such as from 0.25-15 microliters (μL) in volume. Biosensorsmay be implemented using bench-top, portable, and like measurementdevices. Portable measurement devices may be hand-held and allow for theidentification and/or quantification of one or more analytes in asample. Examples of portable measurement devices include the AscensiaBreeze® and Elite® meters of Bayer HealthCare in Tarrytown, N.Y., whileexamples of bench-top measurement devices include the ElectrochemicalWorkstation available from CH Instruments in Austin, Tex. Biosensorsproviding shorter analysis times, while supplying the desired accuracyand/or precision, provide a substantial benefit to the user.

Biosensors may use optical and/or electrochemical methods to analyze thesample. In some optical systems, the analyte concentration is determinedby measuring light that has interacted with or been absorbed by alight-identifiable species, such as the analyte or a reaction or productformed from a chemical indicator reacting with the analyte. In otheroptical systems, a chemical indicator fluoresces or emits light inresponse to the analyte when illuminated by an excitation beam. Thelight may be converted into an electrical output signal, such as currentor potential, which may be similarly processed to the output signal froman electrochemical method. In either optical system, the biosensormeasures and correlates the light with the analyte concentration of thesample.

In electrochemical biosensors, the analyte concentration is determinedfrom an electrical signal generated by an oxidation/reduction or redoxreaction of the analyte or a species responsive to the analyte when aninput signal is applied to the sample. The input signal may be appliedas a single pulse or in multiple pulses, sequences, or cycles. Anoxidoreductase, such as an enzyme or similar species, may be added tothe sample to enhance the electron transfer from a first species to asecond species during the redox reaction. The enzyme or similar speciesmay react with a single analyte, thus providing specificity to a portionof the generated output signal. Examples of some specificoxidoreductases and corresponding analytes are given below in Table I.

TABLE I Oxidoreductase (reagent layer) Analyte Glucose dehydrogenaseβ-glucose Glucose oxidase β-glucose Cholesterol esterase; cholesteroloxidase Cholesterol Lipoprotein lipase; glycerol kinase; glycerol-3-Triglycerides phosphate oxidase Lactate oxidase; lactate dehydrogenase;Lactate diaphorase Pyruvate oxidase Pyruvate Alcohol oxidase AlcoholBilirubin oxidase Bilirubin Uricase Uric acid Glutathione reductaseNAD(P)H Carbon monoxide oxidoreductase Carbon monoxide

A mediator may be used to maintain the oxidation state of the enzyme.Table II, below, provides some conventional combinations of enzymes andmediators for use with specific analytes.

TABLE II Analyte Enzyme Mediator Glucose Glucose Oxidase FerricyanideGlucose Glucose Dehydrogenase Ferricyanide Cholesterol CholesterolOxidase Ferricyanide Lactate Lactate Oxidase Ferricyanide Uric AcidUricase Ferricyanide Alcohol Alcohol Oxidase Phenylenediamine

Electrochemical biosensors usually include a measurement device havingelectrical contacts that connect with electrical conductors in thesensor strip. The conductors may be made from conductive materials, suchas solid metals, metal pastes, conductive carbon, conductive carbonpastes, conductive polymers, and the like. The electrical conductorstypically connect to working, counter, reference, and/or otherelectrodes that extend into a sample reservoir. One or more electricalconductors also may extend into the sample reservoir to providefunctionality not provided by the electrodes.

In many biosensors, the sensor strip may be adapted for use outside,inside, or partially inside a living organism. When used outside aliving organism, a sample of the biological fluid is introduced into asample reservoir in the sensor strip. The sensor strip may be placed inthe measurement device before, after, or during the introduction of thesample for analysis. When inside or partially inside a living organism,the sensor strip may be continually immersed in the sample or the samplemay be intermittently introduced to the strip. The sensor strip mayinclude a reservoir that partially isolates a volume of the sample or beopen to the sample. Similarly, the sample may continuously flow throughthe strip or be interrupted for analysis.

The measurement device applies an input signal through the electricalcontacts to the electrical conductors of the sensor strip. Theelectrical conductors convey the input signal through the electrodesinto the sample present in the sample reservoir. The redox reaction ofthe analyte generates an electrical output signal in response to theinput signal. The electrical output signal from the strip may be acurrent (as generated by amperometry or voltammetry), a potential (asgenerated by potentiometry/galvanometry), or an accumulated charge (asgenerated by coulometry). The measurement device may have the processingcapability to measure and correlate the output signal with the presenceand/or concentration of one or more analytes in the biological fluid.

In conventional amperometry, current is measured during a read pulse asa constant potential (voltage) is applied across the working and counterelectrodes of the sensor strip. The measured current is used to quantifythe analyte in the sample. Amperometry measures the rate at which anelectrochemically active, thus measurable, species is being oxidized orreduced at or near the working electrode. Many variations of theamperometric method for biosensors have been described, for example inU.S. Pat. Nos. 5,620,579; 5,653,863; 6,153,069; and 6,413,411.

A disadvantage of conventional amperometric methods is thenon-steady-state nature of the current after a potential is applied. Therate of current change with respect to time is very fast initially andbecomes slower as the analysis proceeds due to the changing nature ofthe underlying diffusion process. Until the consumption rate of theionized measurable species at the electrode surface equals the diffusionrate, a steady-state current cannot be obtained. Thus, conventionalamperometry methods that measure the current during the transient periodbefore a steady-state condition is reached, may provide more inaccuracythan if the measurement is taken during a steady-state time period.

The measurement performance of a biosensor is defined in terms ofaccuracy and/or precision. Increases in accuracy and/or precisionprovide for an increase in measurement performance for the biosensor.Accuracy may be expressed in terms of bias of the biosensor's analytereading in comparison to a reference analyte reading, with larger biasvalues representing less accuracy, while precision may be expressed interms of the spread or variance among multiple analyte readings inrelation to a mean. Bias is the difference between a value determinedfrom the biosensor and the accepted reference value and may be expressedin terms of “absolute bias” or “relative bias”. Absolute bias may beexpressed in the units of the measurement, such as mg/dL, while relativebias may be expressed as a percentage of the absolute bias value overthe reference value. Reference values may be obtained with a referenceinstrument, such as the YSI 2300 STAT PLUS™ available from YSI Inc.,Yellow Springs, Ohio.

Many biosensors include one or more methods to correct the errorassociated with an analysis. The concentration values obtained from ananalysis with an error may be inaccurate. The ability to correct theseinaccurate analyses may increase the accuracy of the concentrationvalues obtained. An error correction system may compensate for one ormore errors, such as sample hematocrit content, which is different froma reference sample. For example, conventional biosensors may beconfigured to report glucose concentrations presuming a 40% (v/v)hematocrit content for a whole blood sample, regardless of the actualhematocrit content of the sample. In these systems, any glucosemeasurement performed on a whole blood sample containing less or morethan 40% hematocrit will include error or bias attributable to the“hematocrit effect”.

In conventional biosensor sensor strips for determining glucoseconcentrations, glucose may be oxidized by an enzyme, which thentransfers the electron to a mediator. This reduced mediator then travelsto the working electrode where it is electrochemically oxidized. Theamount of mediator being oxidized may be correlated to the currentflowing between the working and counter electrodes of the sensor strip.Quantitatively, the current measured at the working electrode isdirectly proportional to the diffusion coefficient of the mediator. Thehematocrit effect interferes with this process because the red bloodcells block the diffusion of the mediator to the working electrode.Subsequently, the hematocrit effect influences the amount of currentmeasured at the working electrode without any connection to the amountof glucose in the sample.

Hematocrit bias refers to the difference between the reference glucoseconcentration obtained with a reference instrument and an experimentalglucose reading obtained from a biosensor for samples containingdiffering hematocrit levels. The difference between the reference andvalues obtained from the biosensor results from the varying hematocritlevels between specific whole blood samples.

In addition to the hematocrit effect, measurement inaccuracies also mayarise when the measurable species concentration does not correlate withthe analyte concentration. For example, when a sensor system determinesthe concentration of a reduced mediator generated in response to theoxidation of an analyte, any reduced mediator not generated by oxidationof the analyte will lead to the sensor system indicating that moreanalyte is present in the sample than is correct due to mediatorbackground. Thus, “mediator background” is the bias introduced into themeasured analyte concentration attributable to measurable species notresponsive to the underlying analyte concentration.

In an attempt to overcome one or more of these disadvantages,conventional biosensors have attempted multiple techniques, not onlywith regard to the mechanical design of the sensor strip and reagentselection, but also regarding the manner in which the measurement deviceapplies the electric potential to the strip. For example, conventionalmethods of reducing the hematocrit effect for amperometric sensorsinclude the use of filters, as disclosed in U.S. Pat. Nos. 5,708,247 and5,951,836; reversing the polarity of the applied current, as disclosedin WO 2001/57510; and by methods that maximize the inherent resistanceof the sample.

Multiple methods of applying the input signal to the strip, commonlyreferred to as pulse methods, sequences, or cycles, have been used toaddress inaccuracies in the determined analyte concentration. Forexample, in U.S. Pat. No. 4,897,162 the input signal includes acontinuous application of rising and falling voltage potentials that arecomingled to give a triangular-shaped wave. Furthermore, WO 2004/053476and U.S. Pat. Docs. 2003/0178322 and 2003/0113933 describe input signalsthat include the continuous application of rising and falling voltagepotentials that also change polarity.

Other conventional methods combine a specific electrode configurationwith a input signal adapted to that configuration. For example, U.S.Pat. No. 5,942,102 combines the specific electrode configurationprovided by a thin layer cell with a continuous pulse so that thereaction products from the counter electrode arrive at the workingelectrode. This combination is used to drive the reaction until thecurrent change verses time becomes constant, thus reaching a true steadystate condition for the mediator moving between the working and counterelectrodes during the potential step. While each of these methodsbalances various advantages and disadvantages, none are ideal.

As may be seen from the above description, there is an ongoing need forimproved biosensors, especially those that may provide an increasinglyaccurate determination of the analyte concentration in less time. Thesystems, devices, and methods of the present invention overcome at leastone of the disadvantages associated with conventional systems.

SUMMARY

A method for determining the concentration of an analyte in a sample isprovided that includes applying an input signal to the sample, the inputsignal including at least 3 duty cycles within 180 seconds, where eachduty cycle includes an excitation pulse and a relaxation. An outputsignal responsive to a measurable species is measured within 300milliseconds of applying the excitation pulse of at least one of theduty cycles. The concentration of the analyte in the sample isdetermined in response to the measured output signal. The duty cyclesmay each include an excitation at a fixed potential, during which acurrent may be recorded, and a relaxation. The pulse sequence mayinclude a terminal read pulse and may be applied to a sensor stripincluding a diffusion barrier layer. The determined analyteconcentration may include less bias attributable to mediator backgroundthan the same or another method lacking the output signal measurementwithin 300 milliseconds. Through the use of transient current data, theconcentration of the analyte may be determined when a steady-statecondition is not reached during the excitation pulses of the duty cyclesof the input signal. A data treatment may be applied to the measuredcurrents to determine the concentration of the analyte in the sample.

A handheld measurement device adapted to receive a sensor strip isprovided for determining the concentration of an analyte in a sample.The device includes contacts, at least one display, and electroniccircuitry establishing electrical communication between the contacts andthe display. The circuitry includes an electric charger and a processor,where the processor is in electrical communication with a storagemedium. The medium includes computer readable software code, which whenexecuted by the processor, causes the charger to implement an inputsignal including at least 3 duty cycles within 180 seconds between thecontacts. Each duty cycle includes an excitation and a relaxation. Theprocessor is operable to measure at least one current value at the atleast two contacts within 300 milliseconds of the charger applying theexcitation. The processor also is operable to determine the analyte inthe biological fluid in response to the at least one current value.

A biosensor system for determining an analyte concentration in a sampleis provided. The system includes a sensor strip having a sampleinterface adjacent to a reservoir formed by the strip, and a measurementdevice having a processor connected to a sensor interface. The sensorinterface is in electrical communication with the sample interface, andthe processor is in electrical communication with a storage medium. Theprocessor determines an output signal value responsive to theconcentration of the analyte in the sample from the sensor interfacewithin 300 milliseconds of applying an excitation pulse to the sampleinterface. The excitation pulse is part of an input signal including atleast 3 duty cycles within 10 seconds, each duty cycle including anexcitation and a relaxation.

A method of reducing the bias attributable to the hematocrit effect in adetermined concentration of an analyte in a sample is provided thatincludes applying an input signal including at least 3 duty cycleswithin 180 seconds to the sample. The output signal from which theconcentration of the analyte in the sample is determined is recordedwithin 300 milliseconds of applying an excitation pulse.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be better understood with reference to the followingdrawings and description. The components in the figures are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

FIG. 1 represents an electrochemical analytic method for determining thepresence and/or concentration of an analyte in a sample.

FIG. 2 is a graph illustrating the output signals generated from a gatedamperometric input signal.

FIG. 3A shows the hematocrit bias present in analyte concentrationvalues determined from each of the three current values measured fromeach of the seven pulses represented in FIG. 2.

FIG. 3B shows the hematocrit bias span for samples including 50, 100,and 400 mg/dL glucose.

FIG. 4 shows the hematocrit bias for the first and third current valuesfrom P5 in FIG. 3A for multiple whole blood samples.

FIG. 5 depicts a schematic representation of a biosensor that determinesan analyte concentration in a sample.

DETAILED DESCRIPTION

In WO 2007/013915, entitled “Gated Amperometry”, pulsed input signalsare used to analyze analytes in samples. The input signals includealternating excitation and relaxation periods. The present inventionrelates to a system and method of analyzing the output signals from thepulsed input signals to reduce bias, such as that arising from mediatorbackground and the hematocrit effect. By correlating output signalvalues measured within 300 ms of the initiation of an excitation pulse,the accuracy and/or precision of the analysis may be improved.

FIG. 1 represents an electrochemical analysis 100 for determining thepresence and/or concentration of an analyte in a sample. In 110, thesample is introduced to the biosensor. In 120, a portion of the analytein the sample undergoes a redox reaction. In 130, electrons areoptionally transferred from the analyte to a mediator. In 140, ameasurable species is electrochemically excited with an input signal. In150, an output signal is generated and measured. In 160, the sample isallowed to relax, and in 170, additional excitation pulses are input. In180, the presence and/or concentration of the sample is determined fromthe output signal, and in 190, the concentration may be displayed,stored, or the like.

In 110, the sample is introduced to the sensor portion of the biosensor,such as a sensor strip. The sensor strip includes at least one workingand at least one counter electrode. The electrodes may include one ormore reagent layers. The working electrode may include a diffusionbarrier layer that is integral to a reagent layer or that is distinctfrom the reagent layer. When the working electrode includes a distinctdiffusion barrier layer, the reagent layer may or may not be disposed onthe diffusion barrier layer.

A diffusion barrier layer provides a porous space having an internalvolume where a measurable species may reside. The pores of the diffusionbarrier layer may be selected so that the measurable species may diffuseinto the diffusion barrier layer, while physically larger sampleconstituents, such as red blood cells, are substantially excluded.Although conventional sensor strips have used various materials tofilter red blood cells from the surface of the working electrode, adiffusion barrier layer provides an internal porous space to contain andisolate a portion of the measurable species from the sample. A moredetailed treatment of diffusion barrier layers may be found in U.S. Pub.No. 2007/0246357.

In 120 of FIG. 1, a portion of the analyte present in the sample ischemically or biochemically oxidized or reduced, such as by anoxidoreductase. This occurs as the sample hydrates the reagents. Uponoxidation or reduction, electrons optionally may be transferred betweenthe analyte and a mediator in 130. Thus, an ionized measurable speciesis formed, such as from the analyte or a mediator. It may be beneficialto provide an initial time delay, or “incubation period,” for thereagents to react with the analyte. Preferably, the initial time delaymay be from 1 to 10 seconds. A more detailed treatment of initial timedelays may be found in U.S. Pat. Nos. 5,620,579 and 5,653,863.

In 140 of FIG. 1, a measurable species, which may be the charged analytefrom 120 or the charged mediator from 130, is electrochemically excited(oxidized or reduced) with an input signal. Input signals may beelectrical signals, such as current or potential, that pulse or turn onand off at a set sequence. The input signal is a sequence of excitationpulses separated by relaxations. During an amperometric pulse, theelectrical potential applied during the excitation is preferably appliedat a substantially constant voltage and polarity throughout itsduration. This directly contrasts to some conventional excitations wherethe voltage is changed or “swept” through multiple voltage potentialsand/or polarities during data recordation.

During a relaxation of FIG. 1, the electrical signal is off. Offincludes time periods when an electrical signal is not present andpreferably does not include time periods when an electrical signal ispresent but has essentially no amplitude. The electrical signal mayswitch between on and off by closing and opening an electrical circuit,respectively. The electrical circuit may be opened and closedmechanically, electrically, or by other methods.

Input signals may have one or more pulse interval. A pulse interval isthe sum of a pulse and the relaxation constituting a duty cycle. Eachpulse has an amplitude and a width. The amplitude indicates theintensity of the potential, the current, or the like of the electricalsignal. The amplitude may vary or be substantially constant, such asduring amperometry, during the pulse. The pulse width is the timeduration of the pulse. The pulse widths in an input signal may vary orbe substantially the same. Each relaxation has a relaxation width, whichis the time duration of the relaxation. The relaxation widths in aninput signal may vary or be substantially the same.

By adjusting the width of the excitation and relaxation of the dutycycles, gated input signals may increase the accuracy and/or precisionof the analysis. While not wishing to be bound by any particular theory,this increase in accuracy and/or precision may result from drawing themeasurable species excited at the working electrode from the interior ofa diffusion barrier layer. As opposed to measurable species external tothe diffusion barrier layer, which may have a varying rate of diffusiondue to red blood cells and other sample constituents, measurable specieswithin the diffusion barrier layer may have a relatively constantdiffusion rate to the conductor. For example, and as described in U.S.Pub. No. 2007/0246357, entitled “Concentration Determination in aDiffusion Barrier Layer,” a pulse width may be selected to substantiallylimit measurable species excitation to a diffusion barrier layer.

Preferable input signals include at least 3, 4, 6, 8, or 10 duty cyclesapplied during less than 30, 10, or 5 seconds. More preferably, at least3 duty cycles are applied within 10 seconds. Input signals including atleast 4 duty cycles applied in less than 7 seconds are especiallypreferred at present. Preferably, the width of each excitation pulse isindependently selected from between 0.1 and 2 seconds and morepreferably from between 0.2 and 1 second. At present, especiallypreferred input signal pulse widths are independently selected frombetween 0.3 and 0.8 seconds. Preferable pulse intervals are in the rangeof less than 3, 2.5, or 1.5 seconds. At present, input signals havingpulse widths of 0.3 to 0.5 second and pulse intervals from 0.7 to 2seconds are especially preferred. The input signal may have other pulsewidths and intervals.

In 150 of FIG. 1, the biosensor generates an output signal in responseto the measurable species and the input signal. The output signal, suchas one or more current values, may be measured continuously orintermittently and may be recorded as a function of time. Output signalsmay include those that decline initially, those that increase and thendecline, those that reach a steady-state, and those that are transient.Steady-state currents are observed when the current change with respectto time is substantially constant, such as within ±10 or ±5%. Instead ofconventional steady-state or slowly decaying currents, transient(rapidly decaying) current values may be obtained from pulsed inputsignals.

FIG. 2 is a graph illustrating the output signals generated from a gatedamperometric input signal. When plotted as a function of time, eachexcitation pulse results in a transient decay profile having an initialhigh current value that decays. The input signal applied by thebiosensor included eight pulses and seven relaxations, for a total ofseven duty cycles. FIG. 2 omits the first duty cycle and shows that theeighth pulse was not followed by a relaxation. The pulses were appliedat about 200 mV and had a pulse width of about 0.4 seconds. The pulseinterval for the duty cycles was about 1.4 seconds, providing relaxationwidths of about 1 second. The relaxations were provided by an opencircuit. While square-wave pulses were used, other wave types compatiblewith the sensor system and the test sample also may be used.

The biosensor measured the output signal intermittently during eachpulse in FIG. 2 and recorded three current values in a memory device.The output signal values were recorded at about 125 millisecond (ms)intervals starting about 125 ms after the initiation of each pulse. Theintervals between successive recordings may be the same or different. InFIG. 2, three current values from the output signal were recorded andlabeled with the letter l, showing the pulse number and measurementnumber by subscript. Thus, the third current value measured for thefifth pulse is labeled as i_(5,3).

FIG. 3A shows the hematocrit bias present in analyte concentrationvalues determined from each of the three current values measured fromeach of the seven pulses shown in FIG. 2, with larger hematocrit errorrepresented by larger absolute numerical values on the Y-axis. For eachpulse, the first current value showed the least hematocrit bias of thethree values, with the bias difference between the first and thirdvalues becoming larger with each successive pulse. Lower averagehematocrit bias across the measured currents also was observed for eachsuccessive pulse; however, each additional pulse prolonged the length ofthe analysis. Thus, while the current values from P8 included almost nohematocrit error, the first current value from P5 may provide apreferred balance between hematocrit error and analysis time. Also ofinterest is that the first current value measured for P5 had about thesame hematocrit error as the third current value from P8, taken morethan 3 seconds later. These results establish that current valuesmeasured earlier in the pulse width include the least hematocrit error.

FIG. 3B shows the hematocrit bias span for samples including 50, 100,and 400 mg/dL glucose, with larger span values on the Y-axisrepresenting larger hematocrit error. As in FIG. 3A, the first currentvalue showed the least hematocrit bias of the four current valuesmeasured during each pulse, with the bias difference between the firstand fourth values becoming larger with each successive pulse. Theunexpectedly lower hematocrit bias in the first current value measuredfor each pulse was more pronounced at the higher 400 mg/dL glucoseconcentration level. Thus, the accuracy improvement obtained fromcurrent measurements taken early in the decay increased as the glucoseconcentration of the whole blood samples increased.

FIG. 4 shows the hematocrit bias for the first and third current valuesfrom P5 in FIG. 3A for multiple whole blood samples including varyinghematocrit and glucose content. The first current value i_(5,1) showedan R² correlation of 0.18, while the third current value i_(5,3) showedan R² correlation of 0.08, a greater than 50% reduction. The improvedanalyte concentration accuracy obtained from current values takenearlier in the decay is unexpected and directly contrasts with priorteachings that accuracy is achieved from measurements taken in the latersteady-state portion of a decay. These results counterintuitivelyestablish that improved accuracy and/or precision may be obtained frommeasurements taken early in the rapidly changing transient portion ofthe decay.

Preferably, the output current value from which the analyteconcentration is determined is measured within less than 300 ms ofapplying the excitation pulse. More preferably, the output current valueused to determine the analyte concentration of the sample is measuredwithin less than 175 ms from applying an excitation pulse or within 10to 150 ms of applying the pulse. More preferably still, the outputcurrent value from which concentration is determined is measured within30 to 150 ms of applying an excitation pulse. At present, determiningthe concentration of the analyte from an output current value measuredwithin 60 to 150 ms of applying an excitation pulse is especiallypreferred. Preferably, the pulse from which the analytic output currentvalue is measured to determine the concentration of the analyte in thesample is applied within 11 seconds or less of applying the initialexcitation pulse and is more preferably applied within 7 seconds or lessapplying the initial pulse.

In 160 of FIG. 1, the sample undergoes relaxation. The measurementdevice may open the circuit through the sensor strip, thus allowingrelaxation. During the relaxation 160, the current present during theexcitation 140 is substantially reduced by at least one-half, preferablyby an order of magnitude, and more preferably to zero. Preferably, azero current state is provided by an open circuit or other method knownto those of ordinary skill in the art to provide a substantially zerocurrent flow. Preferably, the output signal is not recorded during therelaxation 160.

During the relaxation 160, an ionizing agent, such as an oxidoreductase,may react with the analyte to generate additional measurable specieswithout the effects of an electric potential. For example, a glucosebiosensor including glucose oxidase and a ferricyanide mediator asreagents will produce additional ferrocyanide (reduced mediator)responsive to the analyte concentration of the sample withoutinterference from an electric potential during the relaxation 160.

In 170 of FIG. 1, the biosensor continues to apply pulses from the inputsignal to the working and counter electrodes for the desired timeperiod. The duty cycle including the excitation 140 and the relaxation160 may be repeated or a duty cycle having different pulse widths and/orintervals may be applied.

In 180 of FIG. 1, the biosensor analyzes the output signal valuerecorded within 300 ms of applying a pulse to determine theconcentration of the analyte in the sample. Additional current, time,and/or other values also may be analyzed. In 190, the analyteconcentration value may be displayed, stored for future reference,and/or used for additional calculations.

FIG. 5 depicts a schematic representation of a biosensor 500 thatdetermines an analyte concentration in a sample of a biological fluidusing a pulsed input signal. Biosensor 500 includes a measurement device502 and a sensor strip 504, which may be implemented in any analyticalinstrument, including a bench-top device, a portable or hand-helddevice, or the like. The biosensor 500 may be utilized to determineanalyte concentrations, including those of glucose, uric acid, lactate,cholesterol, bilirubin, and the like. While a particular configurationis shown, the biosensor 500 may have other configurations, includingthose with additional components.

The sensor strip 504 has a base 506 that forms a reservoir 508 and achannel 510 with an opening 1212. The reservoir 508 and the channel 510may be covered by a lid with a vent. The reservoir 508 defines apartially-enclosed volume. The reservoir 508 may contain a compositionthat assists in retaining a liquid sample such as water-swellablepolymers or porous polymer matrices. Reagents may be deposited in thereservoir 508 and/or channel 510. The reagents may include one or moreenzymes, binders, mediators, and like species. The sensor strip 504 alsomay have a sample interface 514 disposed adjacent to the reservoir 508.The sample interface 514 may partially or completely surround thereservoir 508. The sensor strip 504 may have other configurations.

The sample interface 514 has conductors connected to a working electrodeand a counter electrode. The electrodes may be substantially in the sameplane or in more than one plane. Other separation distances between theelectrodes and the lid may be used. The electrodes may be disposed on asurface of the base 506 that forms the reservoir 508. The electrodes mayextend or project into the reservoir 508. A dielectric layer maypartially cover the conductors and/or the electrodes. The sampleinterface 514 may have other electrodes and conductors.

The measurement device 502 includes electrical circuitry 516 connectedto a sensor interface 518 and a display 520. The electrical circuitry516 includes a processor 522 connected to a signal generator 524, anoptional temperature sensor 526, and a storage medium 528.

The signal generator 524 provides an electrical input signal to thesensor interface 518 in response to the processor 522. The electricalinput signal may be transmitted by the sensor interface 518 to thesample interface 514 to apply the electrical input signal to the sampleof the biological fluid. The electrical input signal may be a potentialor current and may be constant, variable, or a combination thereof, suchas when an AC signal is applied with a DC signal offset. The electricalinput signal may be applied as a single pulse or in multiple pulses,sequences, or cycles. The signal generator 524 also may record an outputsignal from the sensor interface as a generator-recorder.

The optional temperature sensor 526 determines the temperature of thesample in the reservoir of the sensor strip 504. The temperature of thesample may be measured, calculated from the output signal, or assumed tobe the same or similar to a measurement of the ambient temperature orthe temperature of a device implementing the biosensor system. Thetemperature may be measured using a thermister, thermometer, or othertemperature sensing device. Other techniques may be used to determinethe sample temperature.

The storage medium 528 may be a magnetic, optical, or semiconductormemory, another storage device, or the like. The storage medium 528 maybe a fixed memory device, a removable memory device, such as a memorycard, remotely accessed, or the like.

The processor 522 implements the analyte analysis and data treatmentusing computer readable software code and data stored in the storagemedium 528. The processor 522 may start the analyte analysis in responseto the presence of the sensor strip 504 at the sensor interface 518, theapplication of a sample to the sensor strip 504, in response to userinput, or the like. The processor 522 directs the signal generator 524to provide the electrical input signal to the sensor interface 518. Theprocessor 522 may receive the sample temperature from the optionaltemperature sensor 526. The processor 522 receives the output signalfrom the sensor interface 518. The output signal is generated inresponse to the redox reaction of the analyte in the sample. Theprocessor 522 measures the output signal within 300 ms of theapplication of an excitation pulse from the signal generator 524. Theoutput signal is correlated with the analyte concentration of the sampleusing one or more correlation equations in the processor 522. Theresults of the analyte analysis may be output to the display 520 and maybe stored in the storage medium 528.

The correlation equations relating analyte concentrations and outputsignals may be represented graphically, mathematically, a combinationthereof, or the like. The correlation equations may be represented by aprogram number (PNA) table, another look-up table, or the like that isstored in the storage medium 528. Instructions regarding implementationof the analyte analysis may be provided by the computer readablesoftware code stored in the storage medium 528. The code may be objectcode or any other code describing or controlling the functionalitydescribed herein. The data from the analyte analysis may be subjected toone or more data treatments, including the determination of decay rates,K constants, ratios, and the like in the processor 522.

The sensor interface 518 has contacts that connect or electricallycommunicate with the conductors in the sample interface 514 of thesensor strip 504. The sensor interface 518 transmits the electricalinput signal from the signal generator 524 through the contacts to theconnectors in the sample interface 514. The sensor interface 518 alsotransmits the output signal from the sample through the contacts to theprocessor 522 and/or signal generator 524.

The display 520 may be analog or digital. The display may be an LCDdisplay adapted to displaying a numerical reading.

In use, a liquid sample for analysis is transferred into the reservoir508 by introducing the liquid to the opening 512. The liquid sampleflows through the channel 510, filling the reservoir 508 while expellingthe previously contained air. The liquid sample chemically reacts withthe reagents deposited in the channel 510 and/or reservoir 508.

The sensor strip 502 is disposed adjacent to the measurement device 502.Adjacent includes positions where the sample interface 514 is inelectrical and/or optical communication with the sensor interface 508.Electrical communication includes the transfer of input and/or outputsignals between contacts in the sensor interface 518 and conductors inthe sample interface 514. Optical communication includes the transfer oflight between an optical portal in the sample interface 514 and adetector in the sensor interface 508. Optical communication alsoincludes the transfer of light between an optical portal in the sampleinterface 502 and a light source in the sensor interface 508.

While various embodiments of the invention have been described, it willbe apparent to those of ordinary skill in the art that other embodimentsand implementations are possible within the scope of the invention.Accordingly, the invention is not to be restricted except in light ofthe attached claims and their equivalents.

1. A method, comprising: applying an input signal to the sample, wherethe input signal has at least 3 duty cycles within 10 seconds, and whereeach duty cycle includes an excitation pulse and a relaxation; measuringan output signal responsive to a measurable species within 300milliseconds of an excitation pulse of at least one duty cycle; anddetermining a concentration of an analyte in a sample in response to themeasured output signal.
 2. The method of claim 1, further comprisingmeasuring the output signal within 300 milliseconds from the start ofthe excitation pulse of the at least one duty cycle.
 3. The method ofclaim 1, where the input signal comprises at least 4 duty cycles within7 seconds.
 4. The method of claim 1, where the pulse width of theexcitation pulse is from 0.1 to 2 seconds.
 5. The method of claim 1,where the pulse width of the excitation pulse is from 0.3 to 0.8seconds.
 6. The method of claim 1, where a pulse interval of at leastone of the duty cycles is less than 3 seconds.
 7. The method of claim 1,where a pulse width of the excitation pulse is from 0.3 to 0.5 seconds,and where a pulse interval of at least one of the duty cycles is from0.7 to 2 seconds.
 8. The method of claim 1, further comprising measuringthe output signal within less than 175 milliseconds of the excitationpulse of the at least one duty cycle.
 9. The method of claim 8, furthercomprising measuring the output signal within less than 175 millisecondsfrom the start of the excitation pulse of the at least one duty cycle.10. The method of claim 1, further comprising measuring the outputsignal within 60 to 150 milliseconds from the start of the excitationpulse of the at least one duty cycle.
 11. The method of claim 1,comprising: measuring an output signal responsive to the measurablespecies within 300 milliseconds of the start of the excitation pulse ofat least one duty cycle applied within 7 seconds of starting an initialexcitation pulse.
 12. The method of claim 1, further comprising:transferring at least one electron from the analyte in the sample to amediator in a sensor strip; and electrochemically exciting themeasurable species in response to the input signal, where the measurablespecies is from at least one of the analyte and the mediator.
 13. Themethod of claim 1, where the excitation pulse has a substantiallyconstant voltage.
 14. The method of claim 1, where the input signalincludes square-wave excitations.
 15. The method of claim 1, furthercomprising determining the concentration of the analyte in the samplewith less bias than a concentration of the analyte determined inresponse to an output signal measured at more than 300 milliseconds fromthe start of the excitation pulse of one of the duty cycles.
 16. Themethod of claim 15, further comprising determining the concentration ofthe analyte in the sample with less bias than a concentration of theanalyte determined in response to an input signal having less than 3duty cycles within 10 seconds.
 17. The method of claim 1, furthercomprising recording at least one current as a function of time duringthe application of the input signal.
 18. The method of claim 1, furthercomprising applying at least one data treatment to at least one currentof the output signal.
 19. The method of claim 1, further comprising:exciting the measurable species internal to a diffusion barrier layer;and substantially excluding from excitation the measurable speciesexternal to the diffusion barrier layer.
 20. The method of claim 1,where the relaxation includes a current reduction to at least one-halfthe current flow at an excitation maxima of the excitation pulse. 21.The method of claim 1, where the relaxation includes a current flowreduction to at least an order of magnitude less than the current flowat an excitation maxima of the excitation pulse.
 22. The method of claim1, where the relaxation includes a substantially zero current flow. 23.The method of claim 1, where the relaxation is at least 0.5 seconds. 24.The method of claim 1, further comprising determining the analyteconcentration from a transient decay, where the output signal includesthe transient decay.
 25. The method of claim 1, where the sample is atleast one of a biological fluid and a derivative of a biological fluid.26. The method of claim 1, where the measuring is performed by aportable measurement device.
 27. A handheld measurement device,comprising: a sensor interface adapted to receive a sensor strip, thesensor interface including contacts; at least one display; andelectronic circuitry establishing electrical communication between thecontacts and the display, where the electronic circuitry includes anelectric charger and a processor in electrical communication, where theprocessor is in electrical communication with a storage medium havingcomputer readable software code, where the processor is operable tocause the electric charger to provide an input signal between thecontacts, where the input signal has at least 3 duty cycles within 10seconds, where each duty cycle includes an excitation pulse and arelaxation, where the processor is operable to measure at least onecurrent value at the at least two contacts within 300 milliseconds ofthe electric charger applying the excitation pulse, and where theprocessor is operable to determine an analyte concentration in abiological fluid or a derivative of a biological fluid in response tothe at least one current value. 28-38. (canceled)
 39. A biosensorsystem, comprising: a sensor strip having a sample interface adjacent toa reservoir formed by the sensor strip; and a measurement device havinga processor connected to a sensor interface, where the sensor interfacehas electrical communication with the sample interface, where theprocessor has electrical communication with a storage medium, where theprocessor determines an output signal value responsive to theconcentration of an analyte in a sample from the sensor interface within300 milliseconds of applying an excitation pulse to the sampleinterface, where the excitation pulse is part of an input signal havingat least 3 duty cycles within 10 seconds, where each duty cycle includesan excitation pulse and a relaxation. 40-51. (canceled)
 52. A method ofreducing bias in a determined concentration of an analyte in a sample,comprising: applying an input signal to the sample, where the inputsignal has at least 3 duty cycles within 10 seconds, and where each dutycycle comprises an excitation pulse and a relaxation; measuring anoutput signal responsive to a measurable species within 300 millisecondsof an excitation pulse of at least one of the duty cycles; anddetermining a concentration of the analyte in the sample in response tothe measured output signal. 53-67. (canceled)